The Pld1Flox allele has loxP sites flanking exons 13-14 (including the catalytic activity-dependent first HKD motif) of the phospholipase D1 gene. Removal of the floxed sequences creates a null allele (see Stock No. 028665). These mice may be useful in studying membrane trafficking, membrane fusion, cancer and neurodegeneration.
Gilbert Di Paolo, Columbia University Medical Center
Phospholipase D1 (Pld1) and its related isoform phospholipase D2 (Pld2) hydrolyze phosphatidylcholine to generate bioactive lipid phosphatidic acid (PA), are implicated in membrane trafficking/membrane fusion, and are elevated/up-regulated in various human carcinomas (including colorectal cancer).
The Pld1Flox allele has loxP sites flanking Pld1 exons 13-14 of the Pld1 gene. Mice homozygous for Pld1Flox are viable and fertile with no reported abnormalities. Following exposure to Cre recombinase, the floxed sequences (including the catalytic activity-dependent first HKD motif) are deleted in the cre-expressing tissues; creating a null allele.
For example, breeding Pld1Flox mice to germline Cre-expressing mice results in the PLD1 global knockout allele Pld1- (available as Stock No. 028665). Those homozygotes (Pld1-/-) are viable and fertile with no reported abnormalities. No protein expression from the knockout allele is detected by western blot analysis of homozygous liver, brain and embryonic fibroblasts.
Furthermore, to study the effect of PLD1-deficiency on spontaneous intestinal tumorigenesis, the Pld1- animals can be bred to ApcMin mice (Stock No. 002020). Specifically, on a C57BL/6J genetic background, PLD1-deficient ApcMin/+ mice have significantly reduced intestinal tumorigenesis and increased survival. The protection is less significant when ApcMin/+ mice are heterozygous for Pld1-. Similar PLD1-deficiency induced protection was observed for an azoxymethane/dextran sodium sulfate (AOM/DSS) mouse model.
In contrast, deficiency of the related isoform PLD2 (Stock No. 028668) does not significantly reduce intestinal tumorigenesis in the C57BL/6J-ApcMin model.
The Pld1Flox allele was created by Dr. Gilbert Di Paolo (Columbia University Medical Center). A targeting vector was designed to have a loxP site upstream of exon 13, and a frt-flanked neo cassette with 3' loxP site just downstream of exon 14 of the phospholipase D1 gene (Pld1) on chromosome 3. The construct was electroporated into C57BL/6 x 129/SvJ embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts, and chimeric males were bred with C57BL/6J females to establish the Pld1Flox Neo colony. The donating investigator reports that mice were then bred to the C57BL/6J-congenic FLP deleter strain (Stock No. 005703) for germline removal of the frt-flanked neo.
The donating investigator reported that the Pld1Flox colony was backcrossed to C57BL/6J wildtype mice for at least six generations (and the Flp-expressing transgene was removed) prior to sending males with black coat color to The Jackson Laboratory Repository in 2016 (see SNP results below). Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).
Of note, the donating investigator reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).
In 2016, a 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the males sent to The Jackson Laboratory Repository. This revealed 6 of 27 markers were not fixed for C57BL/6 allele-type (i.e., still segregating for 129S allele-type markers [6 different chromosomes]). In addition, 3 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating in some mice (note the allele-type for C57BL/6N and 129S are the same for these 5 markers). Collectively, these data show segregation on 7 different chromosomes- suggesting that the mice sent to The Jackson Laboratory Repository were incompletely backcrossed onto C57BL/6, and the C57BL/6 genetic background contributions may be a mix of C57BL/6J;C57BL/6N.
|Allele Name||targeted mutation 1.2, Gilbert Di Paolo|
|Allele Type||Targeted (Conditional ready (e.g. floxed), No functional change)|
|Gene Symbol and Name||Pld1, phospholipase D1|
|Strain of Origin||C57BL/6 x 129/SvJ|
|Molecular Note||A loxP site was inserted upstream of exon 13. An FRT-flanked neo cassette with a 3' loxP site was inserted downstream of exon 14. FLP-mediated recombination removed the neo cassette.|
Mice homozygous for Pld1Flox are viable and fertile with no reported abnormalities. When maintaining a live colony, heterozygous mice may be bred together, to wildtype mice from the colony or to C57BL/6J inbred mice (Stock No. 000664). Alternatively, homozygous mice may be bred together.
When using the Pld1Flox mouse strain in a publication, please cite the originating article(s) and include JAX stock #028664 in your Materials and Methods section.